1. Field of the Invention
The present invention relates to a method of amplifying nucleic acid from a cell using a nonplanar solid substrate.
2. Description of the Related Art
In conventional methods of amplifying nucleic acids from a cell, cell separation, nucleic acid isolation and nucleic acid amplification are separately performed. Generally, cell separation is performed using centrifugal separation and filtration.
Conventional methods of purifying nucleic acids using a solid phase are known. For example, U.S. Pat. No. 5,234,809 discloses a method of purifying nucleic acids using a solid phase to which nucleic acids bind. However, this method is time-consuming and complicated, and thus is not suitable for implementation on a Lab-On-a-Chip (LOC). In addition, this method requires the use of a chaotropic substance. When a chaotropic salt material is not used in the method, the nucleic acids do not bind to the solid phase.
U.S. Pat. No. 6,291,166 discloses a method of archiving nucleic acids using a solid phase matrix. This method is advantageous in that nucleic acids are irreversibly bound to the solid phase matrix, allowing for delayed analysis or repeated analysis of the nucleic acid solid phase matrix complexes. However, in this method, the solid phase matrix has a positively charged surface (e.g., alumina), that must be rendered hydrophilic with basic materials, such as NaOH. Nucleic acids then irreversibly bind to the hydrophilic alumina, and thus cannot be separated from the alumina.
U.S. Pat. No. 5,705,628 discloses a method of reversibly and non-specifically binding DNA from a DNA-containing solution mixed with a salt and polyethylene glycol to a magnetic microparticle having a carboxyl group-coated surface. This method uses a magnetic microparticle having a carboxyl group-coated surface, a salt, and polyethylene glycol, in order to isolate DNA from the solution.
As described above, conventional methods of isolating and purifying a nucleic acid require addition of a high-concentration reagent for DNA binding. However, such addition can affect subsequent processes, such as a polymerase chain reaction (PCR), and therefore cannot be used on a lab-on-a-chip (LOC). In addition, conventional methods of isolating and purifying a nucleic acid are performed independently from a method of purifying or concentrating a cell. Furthermore, a method of performing isolation of a cell from a sample and amplification of nucleic acid from the cell in a single vessel is not known.
Accordingly, there is a need to develop a method of amplifying nucleic acid from a cell using a solid substrate in which a cell is isolated or concentrated by binding the cell to a solid surface, such as a substrate, and immediately thereafter, a nucleic acid derived from the cell can be isolated, purified and concentrated due to high affinity of the solid substrate for the nucleic acid.
In addition, in a conventional method of detecting a microorganism from blood, there are many cases where the concentration of the microorganism is so low that the microorganism cannot be directly detected. To solve this problem, a method of growing the microorganism by culturing the blood has been developed. In general, a medium including sodium polyanethol sulfonate (SPS) is used in blood culture (for example, Blood Culture Bottle (BC); Hy Laboratories Ltd. (Israel)). SPS is a polyanion anticoagulant used for survival of bacterial cells in blood culture. In particular, SPS is added to fluid blood culture media in many laboratories in order to counteract the bacterial inhibitors of fresh human blood. SPS has a structure similar to that of a nucleic acid base, and thus SPS acts as an intercalator which acts to inhibit any subsequent PCR performed on samples obtained from the blood sample. In addition, the SPS is not removed by commercially available nucleic acid isolation kits, such as, for example, a QIAGEN™ kit. Accordingly, for a successful PCR, the SPS has to be separately removed from any nucleic acid which has been isolated using a conventional nucleic acid isolation kit. If the SPS is not removed from a nucleic acid including SPS, for example, nucleic acid isolated from a blood culture including SPS, the sample has to be diluted more than about 5,000 times prior to amplifying the nucleic acid by performing PCR.